EP1608680A2 - Derives de glp-2 - Google Patents

Derives de glp-2

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Publication number
EP1608680A2
EP1608680A2 EP04722534A EP04722534A EP1608680A2 EP 1608680 A2 EP1608680 A2 EP 1608680A2 EP 04722534 A EP04722534 A EP 04722534A EP 04722534 A EP04722534 A EP 04722534A EP 1608680 A2 EP1608680 A2 EP 1608680A2
Authority
EP
European Patent Office
Prior art keywords
glp
pao
amino
propionyl
lys
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP04722534A
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German (de)
English (en)
Inventor
János Tibor KODRA
Nils Langeland Johansen
Lars Thim
Bernd Peschke
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Novo Nordisk AS
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Novo Nordisk AS
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Application filed by Novo Nordisk AS filed Critical Novo Nordisk AS
Publication of EP1608680A2 publication Critical patent/EP1608680A2/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/605—Glucagons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/12—Antidiarrhoeals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/14—Prodigestives, e.g. acids, enzymes, appetite stimulants, antidyspeptics, tonics, antiflatulents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A61P15/06—Antiabortive agents; Labour repressants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00—General protective or antinoxious agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P41/00—Drugs used in surgical methods, e.g. surgery adjuvants for preventing adhesion or for vitreum substitution
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to novel human glucagon-like peptide 2 (GLP-2) peptides and derivatives thereof which have a protracted profile of action.
  • GLP-2 human glucagon-like peptide 2
  • the invention further relates to methods of making and using these GLP-2 peptides and derivatives as well as polynucleotide constructs encoding such GLP-2 peptides and host cells comprising and expressing the GLP-2 peptides, pharmaceutical compositions, uses and methods of treatment.
  • Glucagon-like peptide 2 (GLP-2) is a 33 amino acid residue peptide produced in intestinal L-cells and released following nutrient intake.
  • the amino acid sequence of the human GLP-2 peptide is given in Fig. 1.
  • the GLP-2 peptide is a product of the proglucagon gene.
  • Proglucagon is expressed mainly in the pancreas and the intestine and to some extent in specific neurons located in the brain.
  • the posttranslational processing of proglucagon is however different in pancreas and intestine.
  • proglucagon is processed mainly to Glucagon Related Pancreatic
  • GRPP Polypeptide
  • Glucagon and Major Proglucagon Fragment.
  • GLP-1 Glucagon-Like Peptide 1
  • GLP-2 Glucagon-Like Peptide 2
  • GLP-2 is secreted from the L-cells in the small and large intestine. This secretion is regulated by nutrient intake. The plasma concentration of GLP-2 in normal fasting subjects is around 15 pM increasing to around 60 pM after a mixed meal.
  • GLP-2 The actions of GLP-2 are transduced by a recently cloned glucagon-like peptide-2 receptor.
  • the GLP-2 receptor represents a new member of the G protein-coupled 7TM receptor superfamily.
  • the GLP-2R is expressed in a highly tissue-specific manner predominantly in the gastrointestinal tract and GLP-2R activation is coupled to increased adenylate cyclase activity.
  • Cells expressing the GLP-2R responds to GLP-2, but not to other peptide of the glucagon family (Glucagon, GLP-1 and GIP).
  • GLP-2R has also been reported to be expressed in the brain or more specific the dorsomedial hypothalamic nucleus. This part of the brain is normally thought to be involved in feeding behaviour and it has been shown that GLP-2 inhibits food intake when injected directly into the brain. Induction of intestinal epithelial proliferation by GLP-2 was demonstrated (Drucker, D. J. et al (1996) Proc. Natl. Acad. Sci. USA 93: 7911-7916) and treatment of gastrointestinal deseases by cells grown in medium containing GLP-2 was disclosed (Drucker, D.J and Keneford, J.R., WO 96/32414). WO 97/31943 relates to GLP-2 peptide analogs and the use of certain GLP-2 peptide analogs for appetite suppression or satiety induction.
  • WO 98/08872 Relates to GLP-2 derivatives comprising a lipophilic substituent.
  • WO 96/32414 and WO 97/39031 relates to specific GLP-2 peptide analogs.
  • WO 98/03547 relates to specific GLP-2 peptide analogs, which exhibit antagonist activity
  • GLP-2 peptides and derivatives thereof are useful in the treatment of gastrointestinal disorders.
  • One problem associated with the use of GLP-2 relates to its short biological half-life (about 7 min).
  • the GLP-2 is subject to enzymatic degradation and is rapidly degraded in the plasma via dipeptidylpeptidase IV (DPP-IV) cleavage between residues Ala 2 and Asp 3
  • DPP-IV dipeptidylpeptidase IV
  • It is a further object of the invention to provide a pharmaceutical composition comprising a compound according to the invention and lo use a compound of the invention to provide such a composition.
  • it is an object of the present invention to provide a method of treating gastrointestinal disorders.
  • the protracted profile effect of these new GLP-2 derivatives is achieved by coupling of a GLP-2 peptide to a hydrophilic moiety that results in GLP-2 derivatives with an improved half-life, thereby facilitating the continuous presence of therapeutically effective amount of GLP-2.
  • hydrophilic moieties that result in continuous presence of GLP-2 are covalently attached hydrophilic polymers such as polyethylene glycol and polypropylene glycol that reduce clearance and are not immunogenic.
  • novel modified forms of GLP-2 derivates having specific amino acid residues modified by covalently attaching polyethyleneglycol (PEG).
  • PEG polyethylene glycol
  • PEG polyethylene glycol
  • PEG is a hydrophilic, biocompatible and non-toxic polymer of general formula H(OCH 2 CH 2 ) n OH wherein n>4. Its molecular weight could vary
  • pegylation The in vivo half-life of certain therapeutic proteins and peptides has been increased by conjugating the protein or peptide with PEG, which is termed "pegylation".
  • PEG provides a protective coating and increases the size of the molecule, thus reducing its metabolic degredation and its renal clearance.
  • pegylation has been reported to reduce immunogenicity and toxicity of certain therapeutic proteins. Abuchowski et al. J. Biol. Chem., 252: 3578-3581 (1977).
  • the present invention relates to derivatives of GLP-2 peptides.
  • the derivatives according to the invention have interesting pharmacological properties, in particular they have a more protracted profile of action than the parent GLP-2 peptides.
  • the invention relates to a GLP-2 derivative comprising a GLP-2 peptide, wherein a hydrophilic substituent is attached to one or more amino acid residues at a position relative to the amino acid sequence of SEQ ID NO:1.
  • the invention in a second aspect, relates to a GLP-2 derivative comprising a GLP-2 peptide, wherein a hydrophilic substituent is attached to one or more amino acid residues al a position relative to the amino acid sequence of SEQ ID NO:1 independently selected from the list consisting of D3, S5, S7, D8, E9, M10, N11. T12, 113, L14, D15, N16, L17, A18, R20, D21 , N24, Q28, and D33.
  • the invention in a third aspect, relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a GLP-2 derivative comprising a GLP-2 peptide, wherein a hydrophilic subsliluenl is attached lo one or more amino acid residues at a position relative to the amino acid sequence of SEQ ID NO:1.
  • the invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a GLP-2 derivative comprising a GLP-2 peptide, wherein a hydrophilic substituent is attached to one or more amino acid residues at a position relative to the amino acid sequence of SEQ ID NO:1 independently selected from the list consisting of D3, S5, S7, D8, E9, M10, N11 , T12, 113, L14, D15, N16, L17, A18, R20, D21 , N24, Q28, and D33.
  • a hydrophilic substituent is attached to an amino acid residue at the position D3 relative to the amino acid sequence of SEQ ID NO:1.
  • a hydrophilic substituent is attached to an amino acid residue at the position S5 relative to the amino acid sequence of SEQ ID NO:1. In one embodiment a hydrophilic substituent is attached to an amino acid residue at the position S7 relative to the amino acid sequence of SEQ ID NO:1. In one embodiment a hydrophilic substituent is attached to an amino acid residue at the position D8 relative to the amino acid sequence of SEQ ID NO:1. In one embodiment a hydrophilic substituent is attached to an amino acid residue at the position E9 relative to the amino acid sequence of SEQ ID NO:1. In one embodiment a hydrophilic substituent is attached to an amino acid residue at the position M10 relative to the amino acid sequence of SEQ ID NO:1.
  • a hydrophilic substituent is attached to an amino acid residue at the position N11 relative to the amino acid sequence of SEQ ID NO:1. In one embodiment a hydrophilic substituent is attached to an amino acid residue at the position T12 relative to the amino acid sequence of SEQ ID NO:1. In one embodiment a hydrophilic substituent is attached to an amino acid residue at the position 113 relative to the amino acid sequence of SEQ ID NO:1. In one embodiment a hydrophilic substituent is attached to an amino acid residue at the position L14 relative to the amino acid sequence of SEQ ID NO:1. In one embodiment a hydrophilic substituent is attached to an amino acid residue at the position D15 relative to the amino acid sequence of SEQ ID NO:1.
  • a hydrophilic substituent is attached to an amino acid residue at the position N16 relative to the amino acid sequence of SEQ ID NO:1. In one embodiment a hydrophilic substituent is attached to an amino acid residue at the position L17 relative to the amino acid sequence of SEQ ID NO:1. In one embodiment a hydrophilic substituent is attached lo an amino acid residue at the position A18 relative to the amino acid sequence of SEQ ID NO:1. In one embodiment a hydrophilic substituent is attached to an amino acid residue at the position R20 relative to the amino acid sequence of SEQ ID NO:1. In one embodiment a hydrophilic substituent is attached to an amino acid residue al the position D21 relative to the amino acid sequence of SEQ ID NO:1.
  • a hydrophilic substituent is attached to an amino acid residue at the position N24 relative to the amino acid sequence of SEQ ID NO:1. In one embodiment a hydrophilic substituent is attached to an amino acid residue at the position Q28 relative to the amino acid sequence of SEQ ID NO:1. In one embodiment a hydrophilic substituent is attached to an amino acid residue at the position D33 relative to the amino acid sequence of SEQ ID NO:1. It is to be understood that an amino acid residues at the position relative to the amino acid sequence of SEQ ID NO:1 may be any amino acid residue and not only the amino acid residue naturally present at that position. In one embodiment the hydrophilic substituent is attached to a lysine.
  • the invention relates to the use of a GLP-2 derivative comprising a GLP-2 peptide, wherein a hydrophilic substituent is attached to one or more amino acid residues at a position relative to the amino acid sequence of SEQ ID NO:1 for the preparation of a medicament.
  • the invention relates to the use of a GLP-2 derivative comprising a GLP-2 peptide, wherein a hydrophilic substituent is attached to one or more amino acid residues at a position relative to the amino acid sequence of SEQ ID NO:1 independently selected from the list consisting of D3, S5, S7, D8, E9, M10, N11. T12, 113, L14, D15, N16, L17, A18, R20, D21 , N24, Q28, and D33 for the preparation of a medicament.
  • the invention relates to the use of a GLP-2 derivative comprising a GLP-2 peptide, wherein a hydrophilic substituent is attached to one or more amino acid residues at a position relative to the amino acid sequence of SEQ ID NO:1 for the preparation of a medicament with protracted effect.
  • the invention relates to the use of a GLP-2 derivative comprising a GLP-2 peptide, wherein a hydrophilic substituent is attached to one or more amino acid residues at a position relative to the amino acid sequence of SEQ ID NO:1 independently selected from the list consisting of D3, S5, S7, D8, E9, M10, N11 , T12, 113, L14, D15, N16, L17, A18, R20, D21 , N24, Q28, and D33 for the preparation of a medicament with protracted effect.
  • the invention relates to the use of a GLP-2 derivative comprising a GLP-2 peptide, wherein a hydrophilic substituent is attached lo one or more amino acid residues at a position relative lo the amino acid sequence of SEQ ID NO:1 for the preparation of a medicament for the treatment of intestinal failure or other condition leading to malabsorption of nutrients in the intestine.
  • the invention relates to the use of a GLP-2 derivative comprising a GLP-2 peptide, wherein a hydrophilic substituent is attached lo one or more amino acid residues at a position relative to the amino acid sequence of SEQ ID NO:1 independently selected from the list consisting of D3, S5, S7, D8, E9, M10, N11 , T12, 113, L14, D15, N16, L17, A18, R20, D21 , N24, Q28, and D33 for the preparation of a medicament for the treatment of intestinal failure or other condition leading to malabsorption of nutrients in the intestine.
  • the invention relates to the use of a GLP-2 derivative comprising a GLP-2 peptide, wherein a hydrophilic substituent is attached to one or more amino acid residues at a position relative to the amino acid sequence of SEQ ID NO:1 for the preparation of a medicament for the treatment of small bowel syndrome, Inflammatory bowel syndrome, Crohns disease, colitis including collagen colitis, radiation colitis, post radiation atrophy, non- tropical (gluten intolerance) and tropical sprue, damaged tissue after vascular obstruction or trauma, tourist diarrhea, dehydration, bacteremia, sepsis, anorexia nervosa, damaged tissue after chemotherapy, premature infants, schleroderma, gastritis including atrophic gastritis, postantrectomy atrophic gastritis and helicobacter pylori gastritis, ulcers, enteritis, cul-de-sac, lymphatic obstruction, vascular disease and graft-versus-host, healing after surgical procedures
  • the invention relates to the use of a GLP-2 derivative comprising a GLP-2 peptide, wherein a hydrophilic substituent is attached to one or more amino acid residues at a position relative to the amino acid sequence of SEQ ID NO:1 independently selected from the list consisting of D3, S5, S7, D8, E9, M10, N11 , T12, 113, L14, D15, N16, L17, A18, R20, D21 , N24, Q28, and D33 for the preparation of a medicament for the treatment of small bowel syndrome, Inflammatory bowel syndrome, Crohns disease, colitis including collagen colitis, radiation colitis, post radiation atrophy, non-tropical (gluten intolerance) and tropical sprue, damaged tissue after vascular obstruction or trauma, tourist diarrhea, dehydration, bacteremia, sepsis, anorexia nervosa, damaged tissue after chemotherapy, premature infants, schleroderma, gastritis including atrophic gastriti
  • the invention relates a method for the treatment of insteslinal failure or other condition leading to malabsorption of nutrients in the intestine, the method comprising administering a therapeutically or prophylactically effective amount of a GLP-2 derivative comprising a GLP-2 peptide, wherein a hydrophilic substituent is attached to one or more amino acid residues al a position relative to the amino acid sequence of SEQ ID NO:1 ; to a subject in need thereof.
  • the invention relates a method for the treatment of instestinal failure or other condition leading to malabsorption of nutrients in the intestine, the method comprising administering a therapeutically or prophylactically effective amount of a GLP-2 derivative comprising a GLP-2 peptide, wherein a hydrophilic substituent is attached to one or more amino acid residues at a position relative lo the amino acid sequence of SEQ ID NO:1 independently selected from the list consisting of D3, S5, S7, D8, E9, M10, N11 , T12, 113, L14, D15, N16, L17, A18, R20, D21 , N24, Q28, and D33; to a subject in need thereof.
  • the invention relates a method for the treatment of small bowel syndrome, Inflammatory bowel syndrome, Crohns disease, colitis including collagen colitis, radiation colitis, post radiation atrophy, non-tropical (gluten intolerance) and tropical sprue, damaged tissue after vascular obstruction or trauma, tourist diarrhea, dehydration, bacteremia, sepsis, anorexia nervosa, damaged tissue after chemotherapy, premature infants, schleroderma, gastritis including atrophic gastritis, postantrectomy atrophic gastritis and helicobacter pylori gastritis, ulcers, enteritis, cul-de-sac, lymphatic obstruction, vascular disease and graft-versus-host, healing after surgical procedures, post radiation atrophy and chemotherapy, and osteoporosis, the method comprising administering a therapeutically or prophylactically effective amount of a GLP-2 derivative comprising a GLP-2 peptide, wherein a hydrophilic substituent is
  • the invention relates a method for the treatment of small bowel syndrome, Inflammatory bowel syndrome, Crohns disease, colitis including collagen colitis, radiation colitis, post radiation atrophy, non-tropical (gluten intolerance) and tropical sprue, damaged tissue after vascular obstruction or trauma, tourist diarrhea, dehydration, bacteremia, sepsis, anorexia nervosa, damaged tissue after chemotherapy, premature infants, schleroderma, gastritis including atrophic gastritis, postantrectomy atrophic gastritis and helicobacter pylori gastritis, ulcers, enteritis, cul-de-sac, lymphatic obstruction, vascular disease and graft-versus-host, healing after surgical procedures, post radiation atrophy and chemotherapy, and osteoporosis, the method comprising administering a therapeutically or prophylactically effective amount of a GLP-2 derivative comprising a GLP-2 peptide, wherein a hydrophilic substituent is
  • Short Bowel Syndrome is a devastating clinical condition encountered in a wide spectrum of medical and surgical conditions. The most common causes include irradiation, cancer, mesenteric vascular disease, Crohn's disease and trauma. With improved care of patients with SBS a greater number of patients are surviving for a longer period of time, thus magnifying the need for thera-peutic interventions to reduce or eliminate the long- term problems associated with SBS.
  • SBS patient have up to 7 meals a day they still have problems maintaining normal body weight and these patients are often maintained on parenteral nutrition either at home (HPN) or at the hospital.
  • Chemotherapy (CT) and radiation therapy (RT) for treatment of cancers target rapidly dividing cells.
  • CT/RT tends to produce intestinal mucosal damage as an adverse effect. Gastroenteritis, diarrhea, dehydration and, in some cases, bacteremia and sepsis may ensue.
  • IBD Inflammatory bowel disease
  • Crohn's disease which mainly affects the small intestine
  • ulcerative colitis which mainly occurs in the distal colon and rectum.
  • the pathology of IBD is characterized by chronic inflammation and destruction of the Gl epithelium.
  • Current treatment is directed towards suppression of inflammatory mediators.
  • Stimulation of repair and regeneration of the epithelium by intestinotrophic agents such as GLP-2 derivatives according to the present invention might represent an alternative or adjunct strategy for treatment of IBD.
  • Dextran sulfate (DS)-induced colitis in rodents resembles ulcerative colitis in man, with development of mucosal edema, crypt erosions and abscesses, leading to polyp formation and progression to dysplasia and adenocarcinoma, but the precise mechanism underlying the toxicity of DS is not known.
  • a beneficial effect of GLP-2 peptides in (DS)- induced colitis in mice have been demonstrated, Drucker et al. Am. J. Physiol.276 (Gastrointest. Liver Physiol. 39 ): G79-G91 , 1999.
  • mice receiving 5% DS in the drinking water developed loose blood-streaked stools after 4-5 days and lost 20-25% of their body weight after 9-10 days. Mice that were in addition treated subcutaneously twice daily for the whole period (9-10 days) with either 350 ng or 750 ng A2G-GLP-2(1-33) lost significantly less body weight and appeared much healthier. The effects were dose-dependent. By histology, DS mice treated with A2G-GLP-2(1-33) exhibited a greater proportion of intact mucosal epithelium, increased colon length, crypt depth and mucosal area. These effects were mediated in part via enhanced stimulation of mucosal epithelial cell proliferation.
  • GLP-2 derivatives according to the present invention there is a therapeutic potential for the treatment of IBD of GLP-2 derivatives according to the present invention, potentially in combination with anti-inflammatory drugs.
  • GLP-2 derivatives according to the present invention as an adjunct to anti-inflammatory therapy in IBD.
  • the predominant role of GLP-2 derivatives according to the present invention in IBD would be to enhance the regeneration of compromised intestinal epithelium.
  • GLP-2 peptide as used herein means any protein comprising the amino acid sequence 1-33 of native human GLP-2 (SEQ ID NO: 1) or analogs thereof. This includes but are not limited to native human GLP-2 and analogs thereof.
  • GLP-2 as used herein is intended to include proteins that have the amino acid sequence 1-33 of native human GLP-2 with amino acid sequense of SEQ ID NO:1. It also includes proteins with a slightly modified amino acid sequence, for instance, a modified N-terminal end including N-terminal amino acid deletions or additions so long as those proteins substantially retain the activity of GLP-2. "GLP-2" within the above definition also includes natural allelic variations that may exist and occur from one individual to another. Also, degree and location of glycosylation or other post-translation modifications may vary depending on the chosen host cells and the nature of the host cellular environment.
  • analog or “analogs”, as used herein, is intended to designate a GLP-2 peptide having the sequence of SEQ ID NO:1 , wherein one or more amino acids of the parent GLP-2 protein have been substituted by another amino acid and/or wherein one or more amino acids of the parent GLP-2 protein have been deleted and/or wherein one or more amino acids have been inserted in protein and/or wherein one or more amino acids have been added to the parent GLP-2 protein. Such addition can take place either at the N- terminal end or at the C-terminal end of the parent GLP-2 protein or both.
  • an analog is 70 % identical with the sequence of SEQ ID NO:1. In one embodiment an analog is 80 % identical with the sequence of SEQ ID NO:1. In another embodiment an analog is 90 % identical with the sequence of SEQ ID NO:1. In a further embodiment an analog is 95 % identical with the sequence of SEQ ID NO:1. In a further embodiment an analog is a GLP-2 peptide, wherein a total of up to ten amino acid residues of SEQ ID NO:1 have been exchanged with any amino acid residue.
  • an analog is a GLP-2 peptide, wherein a total of up to five amino acid residues of SEQ ID NO:1 have been exchanged with any amino acid residue. In a further embodiment an analog is a GLP-2 peptide, wherein a total of up to three amino acid residues of SEQ ID NO:1 have been exchanged with any amino acid residue. In a further embodiment an analog is a GLP-2 peptide, wherein a total of up to two amino acid residues of SEQ ID NO:1 have been exchanged with any amino acid residue. In a further embodiment an analog is a GLP-2 peptide, wherein a total of one amino acid residue of SEQ ID NO:1 have been exchanged with any amino acid residue.
  • a fragment thereof means any fragment of the peptide according to formula I or II with at least 15 amino acids. In one embodiment the fragment has at least 20 amino acids. In one embodiment the fragment has at least 25 amino acids. In one embodiment the fragment has at least 30 amino acids. In one embodiment the fragment is according to formula I or II with one amino acid deletion in the C-terminal. In one embodiment the fragment is according to formula I or II with two amino acid deletions in the C-terminal. In one embodiment the fragment is according to formula I or II with three amino acid deletions in the C-terminal. In one embodiment the fragment is according to formula I or II with four amino acid deletions in the C-terminal.
  • the fragment is according lo formula I or II with one amino acid deletion in the N-terminal. In one embodiment the fragment is according lo formula I or II with two amino acid deletions in the N-terminal. In one embodiment the fragment is according lo formula I or II with three amino acid deletions in the N-terminal. In one embodiment the fragment is according lo formula I or II with four amino acid deletions in the N-terminal.
  • derivative is used in the present text to designate a peptide in which one or more of the amino acid residues have been chemically modified, e.g. by alkylation, acylalion, ester formation or amide formation.
  • a GLP derivative is used in the present text to designate a derivative of a
  • GLP-2 peptide In one embodiment the GLP-2 derivative according to the present invention has GLP-2 activity as measured by the ability to bind a GLP-2 receptor (GLP-2R) and/or exert a trophic effects on the small or large intestine.
  • GLP-2R GLP-2 receptor
  • the GLP-2 receptor is selected from the list consisting of rat GLP-2R, mouse GLP-2R and human GLP-2R. It is to be understood, that the hydrophilic substituent is attached to a GLP-2 peptide by covalent attachment.
  • covalent attachment means that the GLP-2 peptide and the hydrophilic substituent is either directly covalently joined to one another, or else is indirectly covalently joined to one another through an intervening moiety or moieties, such as a bridge, spacer, or linkage moiety or moieties.
  • hydrophilic substituent means a radical, which is formally derived from a hydrophilic, water soluble molecule by removal of a hydroxyl-radical, regardless of the actual synthesis chosen.
  • hydrophilic and hydrophobic are generally defined in terms of a partition coefficient P, which is the ratio of the equilibrium concentration of a compound in an organic phase to that in an aqueous phase.
  • a hydrophilic compound has a log P value less than 1.0, typically less than about -0.5, where P is the partition coefficient of the compound between octanol and water, while hydrophobic compounds will generally have a log P greater than about 3.0, typically greater than about 5.0.
  • the polymer molecule is a molecule formed by covalent linkage of two or more monomers wherein none of the monomers is an amino acid residue.
  • Preferred polymers are polymer molecules selected from the group consisting of polyalkylene oxides, including polyalkylene glycol (PAG), such as polyethylene glycol (PEG) and polypropylene glycol (PPG), branched PEGs, polyvinyl alcohol (PVA), polycarboxylate, poly-vinylpyrolidone, polyethylene-co-maleic acid anhydride, polystyrene-co-maleic acid anhydride, and dextran, including carboxymethyl-dextran, PEG being particular preferred.
  • PAG polyalkylene glycol
  • PEG polyethylene glycol
  • PPG polypropylene glycol
  • PVA polyvinyl alcohol
  • PVPVC polycarboxylate
  • poly-vinylpyrolidone polyethylene-co-maleic acid anhydride
  • attachment group is intended to indicate a functional group of the GLP-2 or the GLP-2 derivative capable of attaching a polymer molecule.
  • Useful attachment groups are, for example, amine, hydroxyl, carboxyl, aldehyde, kelone, sulfhydryl, succinimidyl, maleimide, vinylsulfone, oxime or halo acetate.
  • PAO polyalkylene glycol
  • PEG polyethylene glycol
  • PPG polypropylene glycol
  • mPEG methoxypolyethylene glycol
  • the PAO is a polyalkylene glycol (PAG).
  • PAO is a polyethylene glycol (PEG).
  • PAO is a polypropylene glycol (PPG).
  • PPG polypropylene glycol
  • PAO is a branched PEG.
  • PAO is a methoxypolyethylene glycol (mPEG).
  • lore preferred are those compounds where the linker is SBAB or SPAB.
  • treatment is meant to include both prevention of an expected instestinal failure or other condition leading to malabsorption of nutrients in the intestine, such as in post radiation atrophy, and regulation of an already occurring instestinal failure, such as in Inflammatory bowel syndrome, with the purpose of inhibiting or minimising the effect of the condition leading to malabsorption of nutrients in the intestine.
  • Prophylactic administration with the GLP-2 derivative according to the invention is thus included in the term "treatment”.
  • subject as used herein is intended to mean any animal, in particular mammals, such as humans, and may, where appropriate, be used interchangeably with the term “patient”.
  • the macromolecule are covalently attached polymers such as polyethylene glycol or polypropylene glycol; and other hydrophilic macromolecules, e.g. polysaccharides such as dextran, that reduce clearance and are not immunogenic.
  • the polymer molecule to be coupled to the GLP-2 peptide may be any suitable molecule such as natural or synthetic homo-polymer or hetero-polymer, typically with a molecular weight in the range of about 300-100.000 Da, such as about 500-20.000 Da, or about 500-15.000 Da, or 2-15 kDa, or 3-15 kDa, or about 10 kDa.
  • homo-polymers examples include a polyalcohol (i.e., poly-OH), a polyamine (i.e, poly-NH 2 ) and a polycarboxylic acid (i.e., poly-COOH).
  • a hetero-polymer is a polymer comprising different coupling groups such as hydroxyl group and amine group.
  • suitable polymer molecules include polymer molecule selected from the group consisting of polyalkylene oxide, including polyalkylene glycol (PAG), such as polyethylene glycol (PEG) and polypropylene glycol (PPG), branched PEGs, polyvinyl alcohol (PVA), polycarboxylate, poly-vinylpyrolidone, polyethylene-co-maleic acid anhydride, polystyrene-co-maleic acid anhydride, dextran, including carboxymethyl-dextran, or any other polymer suitable for reducing immunicenicity and/or increasing functional in vivo half-life and/or serum half-life.
  • PAG polyalkylene glycol
  • PEG polyethylene glycol
  • PPG polypropylene glycol
  • PVA polyvinyl alcohol
  • polycarboxylate poly-vinylpyrolidone
  • polyethylene-co-maleic acid anhydride polystyrene-co-maleic acid anhydride
  • dextran including carboxymethyl-dextran
  • PEG is the preferred polymer molecule, since it has only a few reactive groups capable of cross-linking compared to e.g. polysaccharides such as dextran.
  • mono-functional PEG e.g., methoxypolyethylene glycol (mPEG) is of interest since its coupling chemistry is relatively simple (only one reactive group is available for conjugating with attachment groups the peptide).
  • the hydroxyl end groups of the polymer molecule must be provided in activated form, i.e. with reactive functional groups (examples of which includes primary amino groups, hydrazide (HZ), thiol (SH), succinate (SUC), succinimidyl succinate (SS), succinimidyl succinamide (SSA), succinimidyl proprionate (SPA), succinimidyl 3-mercaptopropionate (SSPA), Norleucine (NOR), succinimidyl carboxymethylate (SCM), succimidyl butanoate (SBA), succinimidyl carbonate (SC), succinimidyl glutarate (SG), acetaldehyde diethyl acetal (ACET), succinimidy carboxymethylate (SCM), benzotriazole carbonate (BTC), N- hydroxysuccinimide (NHS), aldehyde (ALD), trich
  • reactive functional groups examples of which includes primary amino groups, hydrazide (HZ),
  • Suitable activated polymer molecules are commercially available, e.g. from Nektar, formerly known as Shearwater Polymers, Inc., Huntsville, AL, USA, or from PolyMASC Pharmaceuticals pic, UK or from Enzon pharmaceuticals.
  • the polymer molecules can be activated by conventional methods known in the art, e.g. as disclosed in WO 90/13540. Specific examples of activated linear or branched polymer molecules for use in the present invention are described in the Shearwater Polymers, Inc. 1997 and 2000 Catalogs (Functionalized Biocompatible Polymers for Research and pharmaceuticals, Polyethylene Glycol and Derivatives, incorporated herein by reference).
  • activated PEG polymers include the following linear PEGs: NHS-PEG (e.g. SPA-PEG, SSPA-PEG, SBA-PEG, SS-PEG, SSA-PEG, SC-PEG, SG-PEG, and SCM-PEG), and NOR-PEG, SCM-PEG, BTC-PEG, EPOX-PEG, NCO-PEG, NPC-PEG, CDI-PEG, ALD-PEG, TRES-PEG, VS-PEG, IODO-PEG, IA-PEG, ACET-PEG and MAL- PEG, and branched PEGs such as PEG2-NHS and those disclosed in US 5,672,662, US 5,932,462 and US 5,643,575 both which are incorporated herein by reference.
  • linear PEG e.g. SPA-PEG, SSPA-PEG, SBA-PEG, SS-PEG, SSA-PEG, SC-PEG, SG-
  • the polymer conjugation is designed so as to produce the optimal molecule with respect to the number of polymer molecules attached, the size and form of such molecules (e.g. whether they are linear or branched), and the attachment site(s) on GLP-2 or GLP-2 derivate.
  • the molecular weight of the polymer to be used may e.g., be chosen on the basis of the desired effect to be achieved.
  • the hydrophilic substituent may be attached to an amino group of the GLP-2 moiety by means of a carboxyl group of the hydrophilic substituent which forms an amide bond with an amino group of the amino acid to which it is attached.
  • the hydrophilic substituent may be attached to said amino acid in such a way that an amino group of the hydrophilic substituent forms an amide bond with a carboxyl group of the amino acid.
  • the hydrophilic substituent may be linked to the GLP-2 moiety via an ester bond.
  • the ester can be formed either by reaction between a carboxyl group of the GLP-2 moiety and a hydroxyl group of the substituent-to-be or by reaction between a hydroxyl group of the GLP-2 moiety and a carboxyl group of the substituent-to-be.
  • the hydrophilic substituent can be an alkyl group which is introduced into a primary amino group of the GLP-2 moiety.
  • the GLP-2 derivative comprises a GLP-2 peptide comprising the amino acid sequence of formula II
  • X 2 is Ala, Val or Gly;
  • X 3 is Asp, or Glu;
  • X 5 is Ser, or Lys;
  • X 7 is Ser, or Lys;
  • X 8 is Asp, Glu, or Lys;
  • X 9 is Asp, Glu, or Lys;
  • X 10 is Met, Lys, Leu, lie, or Nor- Leucine;
  • X 11 is Asn, or Lys;
  • X 12 is Thr, or Lys;
  • X 13 is lie, or Lys;
  • X 14 is Leu, or Lys;
  • X 15 is Asp, or Lys;
  • X 16 is Asn, or Lys;
  • X 17 is Leu, or Lys;
  • X 18 is Ala, or Lys;
  • X 20 is Arg, or Lys;
  • X 21 is Asp, or Lys;
  • X 24 is Asn, or Lys;
  • X 28 is Gin,
  • the GLP-2 derivative comprises a GLP-2 peptide GLP-2 peptide comprising the amino acid sequence
  • X 2 is Ala, Val or Gly;
  • X 3 is Asp, or Glu;
  • X 5 is Ser, or Lys;
  • X 7 is Ser, or Lys;
  • X 8 is Asp, Glu, or Lys;
  • X 9 is Asp, Glu, or Lys;
  • X 10 is Met, Lys, Leu, lie, or Nor-Leucine;
  • X 11 is Asn, or Lys;
  • X 12 is Thr, or Lys;
  • X 13 is lie, or Lys;
  • X 14 is Leu, or Lys;
  • X 15 is Asp, or Lys;
  • X 16 is Asn, or Lys;
  • X 17 is Leu, or Lys;
  • X 18 is Ala, or Lys;
  • X 20 is Arg, or Lys;
  • X 21 is Asp, or Lys;
  • X 24 is Asn, or Lys;
  • X 28 is Gin,
  • the GLP-2 derivative comprises a GLP-2 peptide consisting of the amino acid sequence His-X 2 -X 3 -Gly-X 5 -Phe-X 7 -X 8 -X 9 -X 10 -X 11 -X 12 -X 13 -X 14 -X 15 -X 16 -X 17 -X 18 -AIa-X 20 -X 21 -Phe-Ile-X 24 - Trp-Leu-lle-X 28 -Thr-Arg-lle-Thr-X 33
  • X 2 is Ala, Val or Gly;
  • X 3 is Asp, or Glu;
  • X 5 is Ser, or Lys;
  • X 7 is Ser, or Lys;
  • X 8 is Asp, Glu, or Lys;
  • X 9 is Asp, Glu, or Lys;
  • X 10 is Met, Lys, Leu, lie, or Nor- Leucine;
  • X 11 is Asn, or Lys;
  • X 12 is Thr, or Lys;
  • X 13 is lie, or Lys;
  • X 14 is Leu, or Lys;
  • X 15 is Asp, or Lys;
  • X 16 is Asn, or Lys;
  • X 17 is Leu, or Lys;
  • X 18 is Ala, or Lys;
  • X 20 is Arg, or Lys;
  • X 21 is Asp, or Lys;
  • X 24 is Asn, or Lys;
  • X 28 is Gin,
  • X 2 is Ala. In one embodiment X 2 is Gly. In one embodiment X 3 is Asp. In one embodiment X 3 is Glu. In one embodiment X 5 is Ser. In one embodiment X 7 is Ser. In one embodiment X 8 is Asp. In one embodiment X 8 is Glu. In one embodiment X 9 is Asp. In one embodiment X 9 is Glu. In one embodiment X 10 is selected from the group consisting of Met, Leu, lie, and Nor-Leucine. In one embodiment X 11 is Asn. In one embodiment X 12 is Thr. In one embodiment X 13 is lie. In one embodiment X 14 is Leu. In one embodiment X 15 is Asp. In one embod ment X 16 s Asn.
  • X 17 is Leu. In one embodiment X 18 is Ala. In one embod ment X 21 s Asp. In one embodiment X 24 is Asn. In one embodiment X 28 is Gin. In one embod ment X 33 s Asp. In one embodiment X 33 is Glu. In one embodiment X 33 is Asp- Arg.
  • one or more amino acid independently selected from the list consisting of X 5 , X 7 , X 8 , X 9 , X 10 , X 11 , X 12 , X 13 , X 14 , X 15 , X 16 , X 17 , X 18 , X 20 , X 21 , X 24 , X 28 , and X 33 is a Lys.
  • one or more amino acid independently selected from the list consisting of X 5 , X 7 , X 8 , X 9 , X 10 , X 11 , X 12 , X 13 , X 14 , X 15 , X 16 , X 17 , X 18 , X 20 , X 21 , X 24 , X 28 , and X 33 is a Lys.
  • the amino acid X 5 is Lys.
  • the amino acid X 7 is Lys.
  • the amino acid X 8 is Lys.
  • the amino acid X 9 is
  • the amino acid X s Lys In one embodiment the amino acid X s Lys. In one embodiment the amino acid X 11 is Lys In one embodiment the amino acid X s Lys. In one embodiment the amino acid X 13 is Lys In one embodiment the amino acid X s Lys. In one embodiment the amino acid X 15 is Lys In one embodiment the amino acid X s Lys. In one embodiment the amino acid X 17 is Lys In one embodiment the amino acid X s Lys. In one embodiment the amino acid X 20 is Lys In one embodiment the amino acid X 21 s Lys. In one embodiment the amino acid X 24 is Lys In one embodiment the amino acid X 28 s Lys. In one embodiment the amino acid X 33 is Lys In one embodiment the amino acid X ; 33 is Asp-Arg.
  • the GLP-2 peptide is a GLP-2 peptide, wherein a total of up to 5 amino acid residues have been exchanged with any ⁇ -amino acid residue, such as 4 amino acid recidues, 3 amino acid residues, 2 amino acid residues, or 1 amino acid residue.
  • the GLP-2 peptide is selected from the list consisting of: GLP-2(1-33), 34R-GLP-2(1-34), A2G-GLP-2(1-33), A2G/34R-GLP-2(1-34); K30R-GLP-2(1-33); S5K-GLP-2(1-33); S7K-GLP-2(1-33); D8K-GLP-2(1-33); E9K-GLP-2(1- 33); M10K-GLP-2(1-33); N11 K-GLP-2(1-33); T12K-GLP-2(1-33); M3K-GLP-2(1-33); L14K- GLP-2(1-33); D15K-GLP-2(1-33); N16K-GLP-2(1-33); L17K-GLP-2(1-33); A18K-GLP-2(1- 33); D21K-GLP-2(1-33); N24K-GLP-2(1-33); Q28K-GLP-2(1-33); S5
  • the GLP-2 derivative only has one hydrophilic substituent attached to the GLP-2 peptide.
  • the hydrophilic subsliluenl comprises H(OCH 2 CH 2 ) n O- wherein n>4 with a molecular weight from about 200 to about 100.000 daltons.
  • the hydrophilic substituent comprises CH 3 O-
  • the hydrophilic substituent is polyethylen glycol (PEG) with a molecular weight from about 200 to about 5000 Daltons. In one embodiment of the invention the hydrophilic substituent is polyethylen glycol
  • PEG poly(ethylene glycol) with a molecular weight from about 5000 to about 20.000 Daltons.
  • the hydrophilic substituent is polyethylen glycol (PEG) with a molecular weight from about 20.000 to about 100.000 Daltons.
  • the hydrophilic substituent comprises is a methoxy-PEG (mPEG) with a molecular weight from about 200 to about 5000 Daltons. In one embodiment of the invention the hydrophilic substituent is methoxy- polyethylen glycol (mPEG) with a molecular weight from about 5000 to about 20.000 Daltons.
  • the hydrophilic substituent is methoxy- polyethylen glycol (mPEG) with a molecular weight from about 20.000 to about 100.000 daltons.
  • the hydrophilic substituent is attached to an amino acid residue in such a way that a carboxyl group of the hydrophilic substituent forms an amide bond with an amino group of the amino acid residue.
  • the hydrophilic substituent is attached to a Lys residue. In one embodiment of the invention the hydrophilic substituent is attached to an amino acid residue in such a way that an amino group of the hydrophilic substituent forms an amide bond with a carboxyl group of the amino acid residue.
  • the hydrophilic substituent is attached to the GLP- 2 peptide by means of a spacer.
  • the spacer is an unbranched alkane ⁇ , ⁇ - dicarboxylic acid group having from 1 to 7 methylene groups, such as two methylene groups which spacer forms a bridge between an amino group of the GLP-2 peptide and an amino group of the hydrophilic substituent
  • the spacer is an amino acid residue except a Cys residue, or a dipeptide.
  • suitable spacers includes ⁇ -alanine, gamma-aminobutyric acid (GABA), ⁇ -glulamic acid, succinic acid, Lys, Glu or Asp, or a dipeptide such as Gly-Lys.
  • GABA gamma-aminobutyric acid
  • the spacer is succinic acid
  • one carboxyl group thereof may form an amide bond with an amino group of the amino acid residue
  • the other carboxyl group thereof may form an amide bond with an amino group of the hydrophilic substituent .
  • the spacer is Lys, Glu or Asp
  • the carboxyl group thereof may form an amide bond with an amino group of the amino acid residue
  • the amino group thereof may form an amide bond with a carboxyl group of the hydrophilic substituent .
  • a further spacer may in some instances be inserted between the ⁇ -amino group of Lys and the hydrophilic substituent .
  • such a further spacer is succinic acid which forms an amide bond with the ⁇ -amino group of Lys and with an amino group present in the hydrophilic substituent .
  • such a further spacer is Glu or Asp which forms an amide bond with the ⁇ -amino group of Lys and another amide bond with a carboxyl group present in the hydrophilic substituent, that is, the hydrophilic substituent is a N ⁇ -acylated lysine residue.
  • the spacer is selected from the list consisting of ⁇ -alanine, gamma-aminobutyric acid (GABA), ⁇ -glutamic acid, Lys, Asp, Glu, a dipeptide containing Asp, a dipeptide containing Glu, or a dipeptide containing Lys.
  • the spacer is ⁇ -alanine.
  • the spacer is gamma-aminobutyric acid (GABA).
  • the spacer is ⁇ - glutamic acid.
  • a carboxyl group of the parent GLP-2 peptide forms an amide bond with an amino group of a spacer
  • the carboxyl group of the amino acid or dipeptide spacer forms an amide bond with an amino group of the hydrophilic substituent
  • an amino group of the parent GLP-2 peptide forms an amide bond with a carboxylic group of a spacer, and an amino group of the spacer forms an amide bond with a carboxyl group of the hydrophilic substituent.
  • the GLP-2 derivative has one hydrophilic substituent In one embodiment of the invention the GLP-2 derivative has two hydrophilic substituent. In one embodiment of the invention the GLP-2 derivative has three hydrophilic substituent. In one embodiment of the invention the GLP-2 derivative has four hydrophilic substituent. In one embodiment of the invention the GLP-2 derivative is selected from the group consisting of
  • the GLP-2 derivative is selected from the group consisting of
  • H3K (3-(PEG-amino)propionyl)-GLP-2(1-33);
  • A18K(3-(PEG-amino)prop onyl)/K30R-GLP-2(1-33) D21 K(3-(PEG-amino)prop onyl)/K30R-GLP-2(1-33) N24K(3-(PEG-amino)prop onyl)/K30R-GLP-2(1-33) Q28K(3-(PEG-amino)prop onyl)/K30R-GLP-2(1-33)
  • the GLP-2 derivative is selected from the group consisting of
  • the GLP-2 derivative is selected from the group consisting of
  • the GLP-2 derivative is selected from the group consisting of
  • the GLP-2 derivative is selected from the group consisting of
  • hydrophilic group is a sugar moiety.
  • hydrophilic substituent comprises an 2-acetamido-2-deoxy- ⁇ -
  • the GLP-2 derivative is selected from the group consisting of D3E/N16N(2-acetamido-2-deoxy- ⁇ -D-glucopyranosyl)/K30R-GLP-2(1-33)) and D3E/M10L/N11 N(2-acetamido-2-deoxy- ⁇ -D-glucopyranosyl)-GLP-2(1-33).
  • the present invention relates to a GLP-2 derivative in which the C-terminal amino acid residue is present in the form of the amide.
  • the parent GLP-2 peptide can be produced by a method which comprises culturing a host cell containing a DNA sequence encoding the GLP-2 peptide and capable of expressing the GLP-2 peptide in a suitable nutrient medium under conditions permitting the expression of the GLP-2 peptide, after which the resulting GLP-2 peptide is recovered from the culture.
  • the medium used to culture the cells may be any conventional medium suitable for growing the host cells, such as minimal or complex media containing appropriate supplements. Suitable media are available from commercial suppliers or may be prepared according to published recipes (e.g. in catalogues of the American Type Culture Collection).
  • the GLP-2 peptide produced by the cells may then be recovered from the culture medium by conventional procedures including separating the host cells from the medium by centrifugation or filtration, precipitating the proteinaceous components of the supernatant or filtrate by means of a salt, e.g. ammonium sulphate, purification by a variety of chromatographic procedures, e.g. ion exchange chromatography, gelfiltration chromatography, affinity chromatography, or the like, dependent on the type of GLP-2 peptide in question.
  • a salt e.g. ammonium sulphate
  • the DNA sequence encoding the parent GLP-2 peptide may suitably be of genomic or cDNA origin, for instance obtained by preparing a genomic or cDNA library and screening for DNA sequences coding for all or part of the GLP-2 peptide by hybridisation using synthetic oligonucleotide probes in accordance with standard techniques (see, for example, Sambrook, J, Fritsch, EF and Maniatis, T, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York, 1989).
  • the DNA sequence encoding the GLP-2 peptide may also be prepared synthetically by established standard methods, e.g.
  • the DNA sequence may also be prepared by polymerase chain reaction using specific primers, for instance as described in US 4,683,202 or Saiki et al., Science 239 (1988), 487 - 491.
  • the DNA sequence may be inserted into any vector which may conveniently be subjected to recombinant DNA procedures, and the choice of vector will often depend on the host cell into which it is to be introduced.
  • the vector may be an autonomously replicating vector, i.e. a vector which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g. a plasmid.
  • the vector may be one which, when introduced into a host cell, is integrated into the host cell genome and replicated together with the chromosome(s) into which it has been integrated.
  • the vector is preferably an expression vector in which the DNA sequence encoding the GLP-2 peptide is operably linked lo additional segments required for transcription of the DNA, such as a promoter.
  • the promoter may be any DNA sequence which shows transcriptional activity in the host cell of choice and may be derived from genes encoding proteins either homologous or heterologous to the host cell. Examples of suitable promoters for directing the transcription of the DNA encoding the GLP-2 peptide of the invention in a variety of host cells are well known in the art, cf. for instance Sambrook ei al., supra.
  • the DNA sequence encoding the GLP-2 peptide may also, if necessary, be operably connected to a suitable terminator, polyadenylation signals, transcriptional enhancer sequences, and translational enhancer sequences.
  • the recombinant vector of the invention may further comprise a DNA sequence enabling the vector to replicate in the host cell in question.
  • the vector may also comprise a selectable marker, e.g. a gene the product of which complements a defect in the host cell or one which confers resistance to a drug, e.g. ampicillin, kanamycin, tetracyclin, chloramphenicol, neomycin, hygromycin or methotrexate.
  • a selectable marker e.g. a gene the product of which complements a defect in the host cell or one which confers resistance to a drug, e.g. ampicillin, kanamycin, tetracyclin, chloramphenicol, neomycin, hygromycin or methotrexate.
  • a secretory signal sequence (also known as a leader sequence, prepro sequence or pre sequence) may be provided in the recombinant vector.
  • the secretory signal sequence is joined to the DNA sequence encoding the GLP-2 peptide in the correct reading frame.
  • Secretory signal sequences are commonly positioned 5' to the DNA sequence encoding the GLP-2 peptide.
  • the secretory signal sequence may be that normally associated with the GLP-2 peptide or may be from a gene encoding another secreted protein.
  • the host cell into which the DNA sequence or the recombinant vector is introduced may be any cell which is capable of producing the present GLP-2 peptides and includes bacteria, yeast, fungi and higher eukaryotic cells.
  • suitable host cells well known and used in the art are, without limitation, E. coli, Saccharomyces cerevisiae, or mammalian BHK or CHO cell lines.
  • the parent GLP-2 peptide can also be produced using standard methods of solid- phase peptide synthesis technigues.
  • Peptide synthesizers are commercially available from, for example Applied Biosystems in Foster City CA. Reagents for solid phase synthesis are commercially available from, for example Midwest Biotech (Fishers, In).
  • Solid phase peptide synthesizers can be used according to manufactures instruction for blocking interfering groups, protecting the amino acid to be reacted, coupling, decoupling, and capping of unreacted amino acids.
  • an ⁇ - ⁇ /-carbomoyl protected amino acid and an N-terminal amino acid on the growing peptide chain on a resin is coupled at room temperature in an inert solvent such as dimethylformamide.
  • N-methylpyrrolidone or methylenechloride in the present of a coupling reagent such as dicyclohexylcarbodiimide and 1-hydroxybenzotriazole and a base such as diisopropylethylamine.
  • a coupling reagent such as dicyclohexylcarbodiimide and 1-hydroxybenzotriazole
  • a base such as diisopropylethylamine.
  • the ⁇ - ⁇ /-carbomoyl protecting group is removed from the resulting peptide resin using a reagent such as trifluoroacetic acid or piperidine, and the coupling is repeated with the next desired ⁇ /-protected amino acid to be added to the peptide chain.
  • Suitable amine protecting groups are well known in the art and are described, for example in Green and Wuts, "Protecting Groups in Organic Synthesis", John Wiley and Sons, 1999, the entire teaching of which are incorporated by reference. Examples include t-butyloxycarbonyl (tBoc) and fluorenylmethoxycarbonyl (Fmoc).
  • the peptides can be synthesized using standard automated solid-phase synthesis protocols using t-butyloxycarbonyl- or fluorenylmethoxycarbonyl-alpha-amino acids with appropriate side-chain protection.
  • the GLP-2 derivatives of the invention can be prepared by introducing the Polymeric Compounds into the parent GLP-2 peptide using standard methods. Pegylation of peptide and proteins is a well established technique see for example (Roberts et al. Adv. Drug Delivery Rev/. 54: 459-476 (2002)., the contents of which is hereby incorporated in its entirety by reference.
  • N ⁇ -acylation of a Lys residue can be carried out by using an activated amide of the acyl group to be introduced as the acylating agent, e.g. the amide with benzotriazole.
  • the acylation is carried out in a polar solvent in the presence of a base.
  • compositions containing a GLP-2 derivative according to the present invention may be administered parenterally to patients in need of such a treatment.
  • Parenteral administration may be performed by subcutaneous, intramuscular or intravenous injection by means of a syringe, optionally a pen-like syringe.
  • parenteral administration can be performed by means of an infusion pump.
  • a further option is a composition which may be a powder or a liquid for the administration of the GLP-2 derivative in the form of a nasal or pulmonal spray.
  • the GLP-2 derivatives of the invention can also be administered transdermally, e.g. from a patch, optionally a ionlophoretic patch, or Iransmucosally, e.g. bucally.
  • compositions containing a GLP-2 derivative of the present invention may be prepared by conventional techniques, e.g. as described in Remington's Pharmaceutical Sciences. 1985 or in Remington: 77?e Science and Practice of Pharmacy, 19 th edition, 1995.
  • the injectable compositions of the GLP-2 derivati ive of the invention can be prepared using the conventional techniques of the pharmaceuti cal industry which involves dissolving and mixing the ingredients as appropriate to give the des red end product,
  • the GLP-2 derivative s dissolved in an amount of water which is somewhat less than the final volume of the composition to be prepared.
  • An isotonic agent, a preservative and a buffer is added as required and the pH value of the solution is adjusted - if necessary - using an acid, e.g. hydrochloric acid, or a base, e.g. aqueous sodium hydroxide as needed.
  • the volume of the solution is adjusted with water to give the desired concentration of the ingredients.
  • isotonic agents are sodium chloride, mannitol and glycerol.
  • preservatives examples include phenol, m-cresol, methyl p-hydroxybenzoate and benzyl alcohol.
  • solutions containing a GLP-2 derivative according to the present invention may also contain a surfactant in order to improve the solubility and/or the stability of the derivative.
  • a composition for nasal administration of GLP-2 may, for example, be prepared as described in European Patent No. 272097 (to Novo Nordisk A/S) or in WO 93/18785.
  • the GLP-2 derivatives of this invention can be used in the treatment of various diseases.
  • the particular GLP-2 derivative to be used and the optimal dose level for any patient will depend on the disease to be treated and on a variety of factors including the efficacy of the specific peptide derivative employed, the age, body weight, physical activity, and diet of the patient, on a possible combination with other drugs, and on the severity of the case. It is recommended that the dosage of the GLP-2 derivative of this invention be determined for each individual patient by those skilled in the art in a similar way as for known parent GLP-2 peptides.
  • amino acids mentioned herein are L-amino acids.
  • left and right ends of an amino acid sequence of a peptide are, respectively, the N- and C-termini unless otherwise specified.
  • Threonine Thr T A simple system is used herein to describe peptides, fragments, analogs and derivatives of GLP-2.
  • R20K-GLP-2(1-31 ) designates a fragment of GLP-2 formally derived from GLP-2 by deleting the amino acid residues at position 32 and 33 of SEQ ID NO:1 and substituting the naturally occurring amino acid residue arginine at position 20 of SEQ ID NO: 1 by a lysine.
  • R20K(N ⁇ -PEG)/K30R-GLP-2(1 -33) designates a derivative of a GLP-2 peptide analog formally derived from GLP-2 by exchange of the naturally occurring amino acid residue lysine in position 30 of SEQ ID NO:1 with an arginine residue and exchange of the naturally occurring amino acid residue arginine in position 20 of SEQ ID NO:1 with a lysine residue and pegylation of the ⁇ -amino group of the lysine residue in position 20 relative to the amino acid sequence of SEQ ID NO:1.
  • L17K(3-(PEG-amino)propionyl)/K30R-GLP-2(1-33) designates a derivative of a GLP-2 peptide analog formally derived from GLP-2 by exchange of the naturally occurring amino acid residue lysine in position 30 of SEQ ID NO:1 with an arginine residue and exchange of the naturally occurring amino acid residue leucine in position 17 of SEQ ID NO:1 with a lysine residue and pegylation of the ⁇ -amino group of the lysine residue in position 17 relative to the amino acid sequence of formula I by means of the spacer ⁇ - alanine.
  • Several changes in the sequence are separated in the notation by a slash ("/").
  • Derivatized amino acids are noted in a way, in which the moiety it is derivatized with is described in brackets following the one-letter code of the respective amino acid. The specific positions of the derivatization of each of the possible amino acids are defined as shown.
  • Fig. 1 The amino acid sequence of the 33 residues human GLP-2.
  • the N-terminal His-Ala indicates the sequence cleaved of aminopeptidase dipeptidyl peptidase IV during metabolism of GLP-2.
  • the Arg20 and Lys30 residues are the two basic amino acid residues in GLP-2.
  • Fig. 2 List of amino acid sequences used in the detailed description of the invention.
  • the RP-analysis was performed using a Waters 2690 systems fitted with a Waters 996 diode array detector. UV detections were collected at 214, 254, 276, and 301 nm on a 218TP54 4.6 mm x 250 mm 5 ⁇ C-18 silica column (The Seperations Group, Hesperia), which was eluted at 1 ml/min at 42°C. The column was equilibrated with 10% of a 0,5 M ammonium sulfate, which was adjusted to pH 2.5 with 4M sulfuric acid. After injection, the sample was eluted by a gradient of 0% to 60% acetonitrile in the same aqueous buffer during 50 min.
  • Method B1 The Reversed Phase-analysis was performed using a Waters 2690 systems fitted with a Waters 996 diode array detector. UV detections were collected at 214, 254, 276, and 301 nm on a 218TP54 4.6 mm x 250 mm 5 ⁇ C-18 silica column (The Seperations Group, Hesperia), which was eluted at 0,5 ml/min at 42°C. The column was equilibrated with an aqueous solution of TFA in water (0.1%). After injection, the sample was eluted by a gradient of 0% to 60% acetonitrile (+ 0.1 % TFA) in an aqueous solution of TFA in water (0.1 %) during 50 min.
  • UV detections were collected at 214, 254, 276, and 301 nm on a 218TP54 4.6 mm x 250 mm 5 ⁇ C-18 silica column (The Seperations Group, Hesperia), which was eluted at 0,5 ml
  • the Reversed Phase-analysis was performed using a Waters 2690 systems fitted with a Waters 996 diode array detector. UV detections were collected at 214, 254, 276, and 301 nm on a 218TP54 4.6 mm x 250 mm 5 ⁇ C-18 silica column (The Seperations Group, Hesperia), which was eluted at 0,5 ml/min at 42°C. The column was equilibrated with an aqueous solution of TFA in water (0.1%). After injection, the sample was eluted by a gradient of 0% to 90% acetonitrile (+ 0.1% TFA) in an aqueous solution of TFA in water (0.1 %) during 50 min.
  • UV detections were collected at 214, 254, 276, and 301 nm on a 218TP54 4.6 mm x 250 mm 5 ⁇ C-18 silica column (The Seperations Group, Hesperia), which was eluted at 0,5 ml/min at 42°
  • UV detections were collected using a Symmetry C18 , 3.5 um, 3.0 mm x 100 mm column. The elution was performed with a linear gradient of 0-60% of a 0.5% solution of diammonium sulfate in water and 100-40% of water over 15 minutes at a flow-rate of 0.75 ml/min at a temperature of 42°C.
  • Method 02-B4 HPLC (Method 02-B4): The RP-analyses was performed using a Alliance Waters
  • the starting resin (438 mg) used for the synthesis was Fmoc- Asp(OtBu)-Wang resin (Merck Biosciences GmbH, Germany, cat. #: 04-12-2047) with a substitution capacity of 0.57 mmol / g.
  • the protected aminoacid derivatives used were (28)- 6-[1-(4,4-Dimethyl-2,6-dioxo-cyclohexylidene)-ethylamino]-2-(9 H-fluoren-9- ylmethoxycarbonylamino)-hexanoic acid (Fmoc-Lys(Dde)-OH), Fmoc-Arg(Pmc)-OH, Fmoc- Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Ala-OH, Fmoc-lle-OH , Fmoc-Phe-OH, Fmoc-Glu(OtBu)- OH, Fmoc-Gln(Trt)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Asp(OtBu)-OH, Fmoc- Thr(tBu
  • the Dde deprotected resin was suspended in NMP ( 20ml).
  • mPEG-Succinimidyl propionate (mPEG-SPA) (Nektar Therapeutics, CA, USA, cat. #: 2M4M0D0147 ) (6 g, 0.3 mmol.) was added and the suspension was shaken overnight. Then the resin was isolated by filtration and washed extensively with NMP, DCM, 2-propanol, methanol and Et 2 O and dried in vacuo.
  • the resin from 1.c was stirred for 3 h at room temperature, with a mixture of 500 ⁇ l TIS, 500 ⁇ l H 2 O and 20 ml TFA.
  • the resin was removed by filtration and washed with 3 ml TFA.
  • the collected filtrates were concentrated in vacuo. to 5 ml and the crude product was precipitated by addition of 30 ml Et 2 O followed by centrifugation. The pellet was washed with 40 ml Et 2 O two times and then air dried.
  • the peptide D3E/N16N(2-acetamido-2-deoxy-3,4,6-tri-O-acetyl- ⁇ -D- glucopyranosyl)/K30R-GLP-2(1-33) was prepared on a Applied Biosystems 433A Peptide Synthesizer, on a Wang-resin (loading 1.07 mmol/g) applying a standard FMOC strategy using HBTU/HOBT as coupling reagent.
  • the peptide was dissolved in a 5% solution of hydrazine in water (10 ml). This solution was stirred for 1 h. It was diluted with water (10 ml) and purified on a HPLC, using a gradient of 30-60% acetonitrile in water in a 0.1% buffer of TFA. The yield of 1.2 mg was determined by UV absorption at 214 nm assuming an absorption coefficient of 1.7 x 10 6 1 x g "1 xcm "1 .

Abstract

L'invention concerne de nouveaux dérivés de peptides GLP-2 (glucagon-like peptide-2) humains présentant un profil d'action prolongée. L'invention concerne également des compositions pharmaceutiques, des utilisations et des méthodes thérapeutiques.
EP04722534A 2003-03-24 2004-03-23 Derives de glp-2 Withdrawn EP1608680A2 (fr)

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ES2427150T3 (es) 2004-11-01 2013-10-29 Nps Pharmaceuticals, Inc. Tratamiento de pacientes con el síndrome del intestino corto con colon en continuidad
KR101242795B1 (ko) * 2005-05-04 2013-03-12 질랜드 파마 에이/에스 글루카곤 유사 펩티드-2(ｇｌｐ-2) 유사체
EP2051995B1 (fr) * 2006-11-08 2017-02-08 Zealand Pharma A/S Analogues sélectifs du peptide 2 de type glucagon (glp-2)
WO2008116913A2 (fr) * 2007-03-28 2008-10-02 Novo Nordisk A/S Composés peptidiques à pégylation biodégradable transitoire
US20110171164A1 (en) * 2008-09-19 2011-07-14 Nektar Therapeutics Polymer conjugates of glp-2-like peptides
WO2010033240A2 (fr) 2008-09-19 2010-03-25 Nektar Therapeutics Polymères à base de glucide pour administration de médicaments et leurs conjugués
CA2848204C (fr) * 2011-09-12 2023-04-04 Amunix Operating Inc. Compositions de peptide-2 de type glucagon et leurs procedes de fabrication et d'utilisation
CN113710692A (zh) 2019-02-11 2021-11-26 Opko生物科学有限公司 长效glp-2类似物

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US5789379A (en) * 1995-04-14 1998-08-04 Allelix Biopharmaceutical Inc. Glucagon-like peptide-2 analogs
US5994500A (en) * 1996-07-19 1999-11-30 1149336 Ontario Inc. Antagonists of intestinotrophic GLP-2 peptides
WO1998008872A1 (fr) * 1996-08-30 1998-03-05 Novo Nordisk A/S Derives de glp-2
DE60134251D1 (de) * 2000-09-18 2008-07-10 Sanos Bioscience As Verwendung von glp-2-peptiden
EA006160B1 (ru) * 2001-02-16 2005-10-27 Конджачем, Инк. Долгоживущий глюкагоноподобный пептид 2(glp-2, гпп-2) для лечения желудочно-кишечных заболеваний и расстройств
EP1234583A1 (fr) * 2001-02-23 2002-08-28 F. Hoffmann-La Roche Ag Conjugués du PEG et du HGF-NK4
AU2003273300A1 (en) * 2002-09-06 2004-03-29 Bayer Pharmaceuticals Corporation Modified glp-1 receptor agonists and their pharmacological methods of use
US7411039B2 (en) * 2002-10-14 2008-08-12 Novo Nordisk A/S GLP-2 compounds, formulations, and uses thereof

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